Noritaka Nishida

Structure of an integrin with an alphaI domain, complement receptor type 4.

We report the structure of an integrin with an αI domain, αXβ2, the complement receptor type 4. It was earlier expected that a fixed orientation between the αI domain and the β‐propeller domain in which it is inserted would be required for allosteric signal transmission. However, the αI domain is highly flexible, enabling two βI domain conformational states to couple to three αI domain states, and greater accessibility for ligand recognition. Although αXβ2 is bent similarly to integrins that lack αI domains, the terminal domains of the α‐ and β‐legs, calf‐2 and β‐tail, are oriented differently than in αI‐less integrins. Linkers extending to the transmembrane domains are unstructured. Previous mutations in the β2‐tail domain support the importance of extension, rather than a deadbolt, in integrin activation. The locations of further activating mutations and antibody epitopes show the critical role of extension, and conversion from the closed to the open headpiece conformation, in integrin activation. Differences among 10 molecules in crystal lattices provide unprecedented information on interdomain flexibility important for modelling integrin extension and activation.

Structural transitions of complement component C3 and its activation products

Complement sensitizes pathogens for phagocytosis and lysis. We use electron microscopy to examine the structural transitions in the activation of the pivotal protein in the complement pathway, C3. In the cleavage product C3b, the position of the thioester domain moves ≈100 Å, which becomes covalently coupled to antigenic surfaces. In the iC3b fragment, cleavage in an intervening domain creates a long flexible linker between the thioester domain and the macroglobulin domain ring of C3. Studies on two products of nucleophile addition to C3 reveal a structural intermediate in activation, and a final product, in which the anaphylatoxin domain has undergone a remarkable movement through the macroglobulin ring.

Requirement of open headpiece conformation for activation of leukocyte integrin αXβ2

Negative stain electron microscopy (EM) and adhesion assays show that αXβ2 integrin activation requires headpiece opening as well as extension. An extension-inducing Fab to the β2 leg, in combination with representative activating and inhibitory Fabs, were examined for effect on the equilibrium between the open and closed headpiece conformations. The two activating Fabs stabilized the open headpiece conformation. Conversely, two different inhibitory Fabs stabilized the closed headpiece conformation. Adhesion assays revealed that αXβ2 in the extended-open headpiece conformation had high affinity for ligand, whereas both the bent conformation and the extended-closed headpiece conformation represented the low affinity state. Intermediate integrin affinity appears to result not from a single conformational state, but from a mixture of equilibrating conformational states.

Structural basis of the collagen-binding mode of discoidin domain receptor 2

Discoidin domain receptor (DDR) is a cell‐surface receptor tyrosine kinase activated by the binding of its discoidin (DS) domain to fibrillar collagen. Here, we have determined the NMR structure of the DS domain in DDR2 (DDR2‐DS domain), and identified the binding site to fibrillar collagen by transferred cross‐saturation experiments. The DDR2‐DS domain structure adopts a distorted jellyroll fold, consisting of eight β‐strands. The collagen‐binding site is formed at the interloop trench, consisting of charged residues surrounded by hydrophobic residues. The surface profile of the collagen‐binding site suggests that the DDR2‐DS domain recognizes specific sites on fibrillar collagen. This study provides a molecular basis for the collagen‐binding mode of the DDR2‐DS domain.

Simultaneous visualization of the extracellular and cytoplasmic domains of the epidermal growth factor receptor

To our knowledge, no structural study to date has characterized, in an intact receptor, the coupling of conformational change in extracellular domains through a single-pass transmembrane domain to conformational change in cytoplasmic domains. Here we examine such coupling, and its unexpected complexity, using nearly full-length epidermal growth factor receptor (EGFR) and negative-stain EM. The liganded, dimeric EGFR ectodomain can couple both to putatively active, asymmetrically associated kinase dimers and to putatively inactive, symmetrically associated kinase dimers and monomers. Inhibitors that stabilize the active or inactive conformation of the kinase active site, as well as mutations in the kinase dimer interface and a juxtamembrane phosphorylation site, shift the equilibrium among the three kinase association states. This coupling of one conformation of an activated receptor ectodomain to multiple kinase-domain arrangements reveals previously unanticipated complexity in transmembrane signaling and facilitates regulation of receptor function in the juxtamembrane and cytoplasmic environments.

Functional and Structural Stability of the Epidermal Growth Factor Receptor in Detergent Micelles and Phospholipid Nanodiscs

Cellular signaling mediated by the epidermal growth factor receptor (EGFR or ErbB) family of receptor tyrosine kinases plays an important role in regulating normal and oncogenic cellular physiology. While structures of isolated EGFR extracellular domains and intracellular protein tyrosine kinase domains have suggested mechanisms for growth factor-mediated receptor dimerization and allosteric kinase domain activation, understanding how the transmembrane and juxtamembrane domains contribute to transmembrane signaling requires structural studies on intact receptor molecules. In this report, recombinant EGFR constructs containing the extracellular, transmembrane, juxtamembrane, and kinase domains are overexpressed and purified from human embryonic kidney 293 cell cultures. The oligomerization state, overall structure, and functional stability of the purified EGF-bound receptor are characterized in detergent micelles and phospholipid bilayers. In the presence of EGF, catalytically active EGFR dimers can be isolated by gel filtration in dodecyl maltoside. Visualization of the dimeric species by negative stain electron microscopy and single particle averaging reveals an overall structure of the extracellular domain that is similar to previously published crystal structures and is consistent with the C-termini of domain IV being juxtaposed against one another as they enter the transmembrane domain. Although detergent-soluble preparations of EGFR are stable as dimers in the presence of EGF, they exhibit differential functional stability in Triton X-100 versus dodecyl maltoside. Furthermore, the kinase activity can be significantly stabilized by reconstituting purified EGF-bound EGFR dimers in phospholipid nanodiscs or vesicles, suggesting that the environment around the hydrophobic transmembrane and amphipathic juxtamembrane domains is important for stabilizing the tyrosine kinase activity in vitro.

Observation of NMR Signals from Proteins Introduced into Living Mammalian Cells by Reversible Membrane Permeabilization Using a Pore-Forming Toxin, Streptolysin O

We have developed a new in-cell NMR method that is applicable to any type of cell and does not require target protein modification or specialized equipment. The stable-isotope-labeled target protein, thymosin beta4 (Tbeta4), was delivered to 293F cells, which were permeabilized by a pore-forming toxin, streptolysin O, and resealed by Ca(2+) after Tbeta4 uptake. As a result, we successfully observed (1)H-(15)N HSQC signals originating from the Tbeta4, including those from the N-terminal acetylation, which had occurred inside the cell as a post-translational modification.

A pH-regulated dimeric bouquet in the structure of von Willebrand factor

At the acidic pH of the trans‐Golgi and Weibel–Palade bodies (WPBs), but not at the alkaline pH of secretion, the C‐terminal ∼1350 residues of von Willebrand factor (VWF) zip up into an elongated, dimeric bouquet. Six small domains visualized here for the first time between the D4 and cystine‐knot domains form a stem. The A2, A3, and D4 domains form a raceme with three pairs of opposed, large, flower‐like domains. N‐terminal VWF domains mediate helical tubule formation in WPBs and template N‐terminal disulphide linkage between VWF dimers, to form ultralong VWF concatamers. The dimensions we measure in VWF at pH 6.2 and 7.4, and the distance between tubules in nascent WPB, suggest that dimeric bouquets are essential for correct VWF dimer incorporation into growing tubules and to prevent crosslinking between neighbouring tubules. Further insights into the structure of the domains and flexible segments in VWF provide an overall view of VWF structure important for understanding both the biogenesis of ultralong concatamers at acidic pH and flow‐regulated changes in concatamer conformation in plasma at alkaline pH that trigger hemostasis.

A Gel-Encapsulated Bioreactor System for NMR Studies of Protein–Protein Interactions in Living Mammalian Cells†

In-cell NMR spectroscopy is a method used to observe isotopically labeled molecules within living cells. The first in-cell NMR experiment was performed with an E. coli overexpressing a N-labeled protein. For the first application of the in-cell NMR method with eukaryotic cells, isotopically labeled target proteins were introduced, by microinjection, into Xenopus laevis oocytes. Recently, Inomata et al. reported a novel in-cell NMR method utilizing a cell-penetrating tag, which opens the way for the application of in-cell NMR spectroscopy to mammalian cells. Our group also reported an in-cell NMR method for mammalian cells; we used a pore-forming toxin, streptolysin O (SLO), to introduce target proteins by diffusion. By using these methods, protein–drug interactions and intracellular post-translational modifications, such as phosphorylation and acetylation, were successfully detected in vivo. However, the major limitation of the in-cell NMR experiments is the occurrence of cell death during the NMR measurement. As the suspension contains a high density of cells (> 1 10 mL), nutrient depletion occurs rapidly in the anaerobic environment within the NMR tube, thus causing the deterioration of conditions and resulting in cell death during NMR measurements. 9] Therefore, the observation of the NMR signals from proteins in living cells is hampered by the sharp NMR signals derived from the isotopically labeled proteins leaked from the cells. Therefore, in-cell NMR experiments for eukaryotic cells currently have limited applications, such as for obtaining a single NMR spectrum measured within a very short time. Although sparse sampling methods have been utilized to shorten the time required to acquire multidimensional NMR spectra, many existing in vitro NMR experiments that are used to provide information regarding dynamics and protein interactions take several hours to perform. To suppress the cell death during NMR measurements over a longer period of time, we utilized a bioreactor to perfuse the cells in the NMR sample tube. In the early in vivo NMR studies, the perfused cells were entrapped in an agarose gel in a 10 mm NMR tube to sequester them in the signal detection coil, and the P signals of intracellular ATP metabolites in yeast and mammalian cells were observed for a period of several days. 13] In this study, we developed a bioreactor for in-cell NMR spectroscopy (Figure 1); in this

Two-state conformations in the hyaluronan-binding domain regulate CD44 adhesiveness under flow condition

The hyaluronan (HA) receptor CD44 mediates cell adhesion in leukocyte trafficking and tumor metastasis. Our previous nuclear magnetic resonance (NMR) studies revealed that the CD44 hyaluronan-binding domain (HABD) alters its conformation upon HA binding, from the ordered (O) to the partially disordered (PD) conformation. Here, we demonstrate that the HABD undergoes an equilibrium between the O and PD conformations, in either the presence or absence of HA, which explains the seemingly contradictory X-ray and NMR structures of the HA-bound HABD. An HABD mutant that exclusively adopts the PD conformation displayed a higher HA affinity than the wild-type. Rolling of the cells expressing the mutant CD44 was less efficient than those expressing the wild-type, due to the decreased tether frequency and the slow cellular off rate. Considering that the mutant CD44, devoid of the low-affinity state, exhibited impaired rolling, we conclude that the coexistence of the high- and low-affinity states of the HABD is essential for the CD44-mediated rolling.